Quantitative evaluation of sodium butyrate on dual coupling

Quantitative evaluation of sodium butyrate
on dual coupling of human muscarinic receptor M4
S.R. Wang, D. Piwnica, T. Jolas, G. Néliat, B. Fouchaq - Cerep - Le Bois l'Evêque, 86600 Celle l'Evescault, France - www.cerep.com - [email protected]
Methods – 1
120
120
100
80
80
60
40
40
20
0
0
-8
-7
Log conc
-6
-5
-4
-3
� 0 mM
� 1 mM
� 2 mM
� 3 mM
� 5 mM
120
60
20
-12 -11 -10 -9
140
% réponse
100
Oxo + Na Butyrate
Carba + Na Butyrate
100
80
60
40
20
-12 -11 -10 -9
-8
-7
Log conc
-6
-5
-4
-3
0
-12 -11 -10 -9
-8
-7
Log conc
-6
-5
-4
-3
Methods – 2
ÓSequence alignment between amplicon and detected transcript
ÓRNA extraction and cDNA synthesis
RNA was extracted from cultured CHO-S/M4 cells using Trireagent
(Sigma), 1.0 µg of total RNA was reversely transcripted to cDNA using
MMLV and random hexamers (Clontech).
ÓReal-time PCR on IQ5 cycler
Alignment between amplicon of CHO/M4 and NM_000741
0B&+2BDPSOLFRQ
10B25)B
0B&+2BDPSOLFRQ
10B25)B
Real-time PCR was performed by SYBR Green technology using Bio-Rad
Master mix.
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The condition of PCR for each pair of primer was optimized by temperature
gradient amplification.
n
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Gene
Primer
name
Qiagen
number
Detected
transcript
Amplicon
size
M4 receptor
Hs-CHRM4
QT00214963
NM_000741
142 bp
G ai 3
Mm-Gnai3
QT01062278
NM_010306
109 bp
G as
Hs-Gnas
QT00021315
NM_000516
91bp
Acknowledgements
GAPDH
Mm-Gapd
QT00309099
NM_001001303
100 bp
We acknowledge Mrs Karine Cheroux,
Ms Maryse Martin and Céline Billy for technical
assistance. We thank Mrs Catherine Moreau and
Mr Loïc Dorgeret for poster preparation.
18S ribosome
Mm-Rn18S
QT01036875
X00686
149 bp
18S/28S ribosome
Rn-Rn1-1
QT00199374
M11188
103 bp
Cyclophilin A
Hs-PPIA
QT00052311
NM_021130
121 bp
Mm = mouse Rn = rat
Hs = human
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0.6
0.4
0.2
0
GAPDH
PPIA
18S
18S & 28S
Data are
representative of three
experiments.
Standard curve
25
y = -3.5096x + 28.593
R2 = 0.9983
E = 92.7%
20
15
Dose-effect
of sodium butyrate
on the expression
copy number of
G ai3
-16
-15
-14
-13
-11
-12
Data are
representative of three
experiments.
Standard curve G ai3
25
slope -3.32 ± 0.0321
R2 = 0.999
E = 10-(1/S) -1
E = 101%
20
15
10
Dose-effect
of sodium butyrate
on the expression
copy number of
G as
-16
-15
-14
[plasmid] g/µl
-13
-12
Data are shown for
mean ± S.D for three
experiments each
performed in triplicate
slope -3.56 ± 0.0977
R2 = 0,993
E =10-(1/S) -1
E = 90.9%
30
25
15
10
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-16
-15
-14
-13
[plasmid] g/µl
-12
-11
Data are shown for
mean ± S.D for three
experiments each
performed in triplicate
15
CHO-s/M4
CHO-s/M4 + 1 mM Na butyrate
CHO-s/M4 + 2 mM Na butyrate
CHO-s/M4 + 3 mM Na butyrate
CHO-s/M4 + 5 mM Na butyrate
12
9
6
3
0
M4
Standard curve G as
35
20
fold change expression
(2-∆∆ct)
0.8
Dose-effect
of sodium butyrate
on the expression
of M4 receptor,
G ai3 and G as
(housekeeping
gene 18S
ribosome)
copy number / ng RNA
fold change of expression
1.0
5
Gαi3
GαS
40k
35k
30k
25k
20k
15k
10k
5k
0
0mM
1mM
0mM
1mM
0mM
1mM
2mM
3mM
5mM
2mM
3mM
5mM
2mM
3mM
5mM
sodium butyrate
400
350
300
250
200
150
100
50
0
sodium butyrate
400K
350K
300K
250K
200K
150K
100K
50K
0
sodium butyrate
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nConclusion
n
Our results showed for the first time that the expression copy number of G αs protein is much more important
than G αi3 (G αi3 is predominantly expressed in CHO cells, data not shown).
n
Dose
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M4 receptor dual coupling (Gαi/Gαs) is dependent on the expressed receptor density and the agonist concentration.
n
A
n
Use of sodium butyrate to manipulate the expression level of heterologous receptor and G protein might provide
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dependant enhancement of sodium butyrate on the expression of M4 receptor (10 to 12 fold at 5 mM)
was observed. In contrast the expression of G αi3 and G αs genes was slightly affected by sodium butyrate
treatment.
No agonist trafficking was observed.
Alignment between amplicon of CHO/G aS and NM_00516
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*DOSKD6B10
� 5.0 mM SB
10
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Alignment between amplicon of CHO/G ai3 and NM_010306
*DOSKDLB&+2BDP
*DOSKDLB10
ÓPrimers used in real-time PCR (Qiagen)
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Dose-effect
of sodium butyrate
on the expression
copy number of
M4 receptor
� 0.0 mM SB
copy number / ng RNA
Standard curves
n Exact quantification of the DNA content for each plasmid
was done in various dilution and repeats on a spectrophotometer.
n Six 10-fold serial dilutions were made starting from a plasmid
concentration of 10-10g/µl.
n A standard curve was drawn by plotting the threshold cycle
(Ct) against the log of the concentration of each plasmid.
n The copy number (per µl) was calculated as follows:
(6 x1023) x ADN (g/µl)/ plasmid size (bp)x 660 (g/mol)
Dose-effect of sodium butyrate on the dual coupling of M4 receptor activated by different agonists (HTRF® cAMP assay)
Ach + Na Butyrate
Plasmid construction
n Conventional PCR was performed to generate gene-specific
amplicon using the same primers necessary for real-time PCR.
n The purified PCR products were cloned to T/A cloning
pCR2.1 vector (Invitrogen). The amplicon identity was confirmed by sequencing.
GAPDH,
Cyclophilin A
(PPIA), 18S
ribosome and
18S/28S ribosome
1.2
copy number / ng RNA
In the present study, comparative
and absolute real-time PCR were
used to evaluate the effect of
sodium butyrate on the expression
of endogenous G proteins and a
heterologous receptor in CHO-S
M4 cells.
CHO-S M4 cells were harvested by EDTA and re-suspended in HBSS buffer containing 0.1% glucose
and 0.5 mM IBMX. The cells were dispensed into a black 96 half-well plate at a density of 10,000
– 15,000 cells per well. After 30 min incubation at room temperature, an equal volume of assay
buffer containing 3.0 µM forskolin, then acetylcholine, oxotremorine or carbachol were added. After
30 min incubation cAMP was measured via the HTRF® (homogeneous time-resolved fluorescence)
technology (CisBio International).
% réponse
Muscarinic receptor M4 has been
largely reported as a G αi and
Gααs dual coupling receptor.
In our study, a biphasic cAMP
response induced by acetylcholine,
oxotremorine and carbachol,
was demonstrated in sodium
butyrate treated CHO-S M4 cells
in a dose dependent manner.
Sodium butyrate is known as a
gene expression modulator. This
intriguing observation suggests
that stoichiometry state of gene
expression between receptor and
G proteins could be responsible for
G αi and G αs dual coupling.
ÓAbsolute real-time PCR
ÓcAMP assay
% réponse
Functional screening of GPCR targeted compounds is becoming a
preferred primary step in the drug
discovery process. However, it is
challenging to obtain a workable
robust assay for G αi/o coupled
receptors. Several factors may explain this phenomena, one being
G αi/ G αs dual coupling.
2-∆∆Ct method was used to determine the relative abundance
of a studied gene expression between the control and the
sodium butyrate treated cells. ∆Ct is the difference in Ct
values between housekeeping (used for normalisation) and
specific gene.
Effect of sodium
butyrate (SB)
on the expression
of housekeeping
genes –
Ct
The human genomic DNA (Clonetech) was used to isolate the gene encoding human M4 receptor
(PCR with specific primers). The corresponding gene was cloned into a mammalian expression vector
pCI/neo (Promega) and then stably transfected into CHO-S cells (Invitrogen).
n CHO-S M
4 cells were cultured in DMEM with 5% dialyzed FCS and G418. 1 mM to 5 mM of
sodium butyrate (Sigma) were added 12-18 h prior to experimentation.
n
ÓRelative real-time PCR
Ct
ÓExpression of recombinant human muscarinic 4 receptor in CHO-S cells
Ct
nIntroduction
Methods – 3
high stoichiometry between G αs and M4 receptor gene in sodium butyrate untreated CHO-S M4 cells does
not favor the M4/ G αs coupling at the 10 pM to 1 µM concentration range of agonists, therefore, physiological
pleiotropic G protein coupling could not exist for M4 receptor.
a new approach in the development of a robust functional assay for G αi coupling receptor.